Hepatitis C virus (HCV) is estimated to infect at approximately 170 million people worldwide, and is responsible for chronic liver disease and increased risk of cirrhosis and hepatocellular carcinoma. Treatment of HCV is principally limited to antiviral regimens, the efficacies of which are largely influenced by several biological parameters, such as the virus genotype. HCV genotyping has therefore been widely used to predict the response to antiviral therapy and to optimize the duration of treatment. HCV genotyping has also become an essential tool for epidemiological studies and for tracing sources of contamination by HCV.
The plus-strand HCV RNA genome is approximately 9600 nucleotides in length and encodes at least one open-reading frame with approximately 3010 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce structural and non-structural (NS) proteins. HCV isolates are characterized by a high degree of genetic variability due to the lack of fidelity of the HCV RNA-dependent RNA polymerase, which is encoded by the non-structural 5B (NS5B) gene. In addition, as a result of endogenous mutation or infection by a plurality of species, also gives rise to genetically variable quasi-species of HCV within a single patient. Six main genotypes of HCV, and over a hundred subtypes, have been described.
The genetic variability of HCV complicates the processes of amplification, sequencing, and genotyping. These processes typically rely upon use of oligonucleotide primers and probes (e.g., PCR amplification primers, sequencing primers, and site-specific probes) that are complementary to and are capable of hybridizing to corresponding nucleic acid sequences of the HCV genome. As a result of the high degree of variability of the HCV genome, oligonucleotide primers and probes complementary to one species of HCV may not be complementary to another species. Such primers and probes must therefore be designed for specificity to highly conserved regions. Alternatively, assays must use mixtures of degenerate primers and probes that are complementary to all species.
Some genotyping methods have focused on the 5′ noncoding (5′NC) region. The 5′ non-coding (NC) region of HCV is highly conserved, yet contains type-specific polymorphisms that can be utilized to distinguish between genotypes. To date, most of the commercially available 5′NC region genotyping assays have been based on PCR amplification and fragment analysis by RFLP or hybridization to oligonucleotide probes. These types of assay are rapid but not as accurate as sequencing-based assays. For this reason, alternative genomic regions have been proposed for use in genotyping HCV, including the NS5B region.
The most accurate and direct method of genotyping HCV is to sequence the virus genome in a region that is sufficiently divergent among various species to distinguish between virus types and subtypes. Equally importantly, databases for phylogenetic analysis must be readily available to analyze the sequences generated from these regions.
Commercially available sequencing-based HCV genotyping assays include, for example, the TRUGENE HCV 5′NC Genotyping Kit (Bayer HealthCare), which is a rapid sequencing-based assay utilizing the 5′NC region of HCV. Sequence data generated by this assay are directly analyzed utilizing a phylogenetic 5′NC region database (TRUGENE HCV 5′NC software module v3.1.1). Previously, 5′NC databases have included sequences from various sources that have never been fully validated and, in some cases, subtype assignments for particular strains have been discordant when 5′NC or NS5B sequences were analyzed.
There is a continuing need to improve sequencing-based HCV assays, so as to improve identification of HCV types and subtypes for purposes of clinical analysis and therapeutic intervention.